Team:TU-Delft/PeptideCharacterization
From 2013.igem.org
![](https://static.igem.org/mediawiki/2013/e/e4/Frog_transparent.png)
Peptide Characterization
An important part of the project is inhibition of growth or killing of bacteria with the use of antimicrobial peptides (AMPs). In order to get an idea of the toxicity of our peptides we conducted several minimal inhibiting concentration (MIC) experiments. These MIC measurements where done on E. coli, S. delphini and B. subtilis as respectively representatives for our expression host, the Staphylococcus family and Gram-positives. The AMPs Signiferin, maximin H5 and magainin were chosen from literature [1,2,3] as being active against staphylococcus species but not against E. coli in order to able to use the latter as the expression host.
Procedure
- Grow the appropriate strains overnight and make a 1/50 to 1/100 dilution the next morning and wait for the OD at 600nm to reach 0,4-0,6.
- Make a 100µM stock of the AMP in LB medium, this solution will serve as a 100 X stock, as the expected MIC range of our AMPs lays between 5 and 40 µM.
- Then make the combinations as shown in table 1 in a 96 well plate, for the plate reader to take readings using an plate reader capable of shaking and heating to 37˚C. Taking measurements every 10 minutes.
No cells + 1mM | 40µM | 30µM | 25µM | 20µM | 15µM | 10µM | 7.5µM | 5.0µM | 2.0µM | 1.0µM | Cell + No AMP | |
LB(µL) | 90 | 55 | 65 | 70 | 91 | 75 | 80 | 85 | 87.5 | 90 | 93 | 94 |
Cells(µL) | - | 5 | 5 | 5 | 5 | 5 | 5 | 5 | 5 | 5 | 5 | 5 |
100μΜ(µL) | 10 | 40 | 30 | 25 | 20 | 15 | 10 | 7.5 | 5.0 | 2.0 | 1.0 | - |
Results
Signifirin MIC determination
The MIC of signifirin on E.coli, B. subtilis and S. delphini has been done according to the protocol described above (fig. 1). S. delphini is the most sensitive for signiferin, as the MIC was determined to be 1µM (fig. 1A), no growth is seen at this concentration.
The expected selectivity for Staphylococcus of the chosen peptide was confirmed by these experiments, as for all the peptides for which we could determine a MIC, that MIC was the lowest for S. delphini. Maximin H5 and magainin did not give a measurable reduction in growth below 40µM, making us decide not to proceed testing, as modeling showed it was not possible to reach these concentrations through expression in E.coli {link to modeling page} .
Of the novel peptides, designed as described ((here), link), two were determined to be active against S. delphini; sebastini and joepini with MICs of respectively 30 and 40µM.
![](https://static.igem.org/mediawiki/2013/d/d2/Figure1MIC.png)
Figure 1: MICs of Maximim-H5
![](https://static.igem.org/mediawiki/2013/c/cb/Figure2MIC.jpg)
Figure 2: MICs of Maximim-H5 and Magainin
![](https://static.igem.org/mediawiki/2013/e/ef/Coli_high_conc.jpg)
Figure 3: MICs of Maximim-H5 and Magainin